Fibrous protein fusions and use thereof in the formation of advanced organic/inorganic composite materials

ABSTRACT

The claimed invention provides a fusion polypeptide comprising a fibrous protein domain and a mineralization domain. The fusion is used to form an organic-inorganic composite. These organic-inorganic composites can be constructed from the nano- to the macro-scale depending on the size of the fibrous protein fusion domain used. In one embodiment, the composites can also be loaded with other compounds (e.g., dyes, drugs, enzymes) depending on the goal for the materials, to further enhance function. This can be achieved during assembly of the material or during the mineralization step in materials formation.

CROSS REFERENCED APPLICATIONS

This application is a continuation of U.S. Ser. No. 13/347,801 filedJan. 11, 2012, which is a continuation of U.S. Ser. No. 13/097,538 filedApr. 29, 2011, now U.S. Pat. No. 8,129,141, issued Mar. 6, 2012, whichis a continuation of U.S. Ser. No. 11/794,934 filed on Jan. 14, 2008,now U.S. Pat. No. 7,960,509, issued Jun. 14, 2011, which is a 371National Phase Entry Application of International Application No.PCT/US2006/01536, filed Jan. 17, 2006, which designates the U.S., andwhich claims the benefit under 35 U.S.C. §119(e) of U.S. ProvisionalPatent Application No. 60/644,264, filed Jan. 14, 2005, the contents ofall of which are incorporated herein by reference in their entireties.

GOVERNMENT SUPPORT

This invention was supported by FA9550041-0363 awarded by the U.S. AirForce and EB003210-01 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

Many biomedical procedures require the provision of healthy tissue tocounteract the disease process or trauma being treated. This work isoften hampered by the tremendous shortage of tissues available fortransplantation and/or grafting. Tissue engineering may ultimatelyprovide alternatives to whole organ or tissue transplantation. In orderto generate engineered tissues, various combinations of biomaterials andliving cells are currently being investigated. Although attention isoften focused on the cellular aspects of the engineering process, thedesign characteristics of the biomaterials also constitute a majorchallenge in this field.

In recent years, the ability to regenerate tissues and to control theproperties of the regenerated tissue have been investigated by trying tospecifically tune the mechanical or chemical properties of thebiomaterial scaffold (Kim et al., 1997; Kohn et al. 1997). The majorityof work has involved the incorporation of chemical factors into thematerial during processing, or the tuning of mechanical properties byaltering the constituents of the material.

The foregoing methods have been used in an attempt to utilize chemicalor mechanical signaling to affect changes in the proliferation and/ordifferentiation of cells during tissue regeneration. Despite suchefforts there remains in the art a need for improved biomaterials,including composite materials, particularly those with a better capacityto support complex tissue growth in vitro (in cell culture) and in vivo(upon implantation).

SUMMARY OF THE INVENTION

The claimed invention provides a fusion polypeptide comprising a fibrousprotein domain and a mineralization domain.

In one embodiment the fibrous protein domain is obtained from silk,collagens, coiled-coiled leucine zipper proteins, elastins, keratins,actins, and tubulins.

For example, in one preferred embodiment, the fibrous protein domaincomprises an amino acid sequence from the silk protein Spidroin 1, suchas the fibrous protein domains indicated by (SEQ ID NO: 1) or (SEQ IDNO: 3), which are derived from Spiroidin 1 of Nephila clavipes. Arecombinant fibrous protein domain can be generated, which has multiplerepeats of a fibrous protein domain.

In one embodiment, the fibrous domain sequence of (SEQ ID NO: 1) isrepeated 15 times throughout the fibrous protein domain, which is usedin the fusion proteins of the invention, i.e., the fibrous proteindomain sequence indicated by (SEQ ID NO: 4), referred to herein as15mer.

In one embodiment, a fibrous domain sequence of (SEQ ID NO: 1) isrepeated 15 times throughout the fibrous protein domain that is used inthe fusion proteins of the invention and has a CRGD4 (SEQ ID NO: 2)linker sequence, i.e. the fibrous protein domain sequence indicated by(SEQ ID NO: 5), referred to herein as CRGD-15mer.

In one embodiment, the fusion polypeptide of the invention has amineralizing domain that is capable of inducing the formation ofhydroxyapatite, silica, cadmium sulfide or magnetite.

In one embodiment, the mineralization domain is obtained from dentinmatrix protein 1 (DMP1), bone sialoprotein (BSP), or silaffin-1 (Sil1)protein.

In one embodiment, the mineralization domain is derived from dentinmatrix protein 1 (DMP1) and is (SEQ ID NO: 6) or (SEQ ID NO: 7).

In one embodiment, the mineralization domain is derived from bonesialoprotein (BSP) and is (SEQ ID NO: 8).

In one embodiment, the mineralizing domain is selected from the groupconsisting of the 19 amino-acid R5 peptide of the Sil1 protein (SEQ IDNO: 17), the R2 peptide of the Sil1 protein (SEQ ID NO: 18), the 19amino-acid R3 peptide of the Sil1 protein (SEQ ID NO: 19), the 19amino-acid R6 peptide of the Sil1 protein (SEQ ID NO: 20) and the 15amino-acid R1 peptide of the Sil1 protein (SEQ ID NO: 21).

The invention also provides for the following fusion polypeptides thatcomprise a fibrous protein domain and a mineralization domain: thefusion polypeptide (SEQ ID NO: 9), the fusion polypeptide (SEQ ID NO:10), the fusion polypeptide (SEQ ID NO: 11), the fusion polypeptide (SEQID NO: 12), a fusion polypeptide comprising a fusion of the 15 mer silkfibrous protein domain (SEQ ID NO: 4) and the R5 peptide of the Sil1protein (SEQ ID NO: 17) and a fusion polypeptide comprising a fusion ofthe CRGD-15 mer silk fibrous protein domain (SEQ ID NO: 5) and the R5peptide of the Sil1 protein (SEQ ID NO: 17).

A method for forming a fibrous protein inorganic-composite material isalso provided. The method comprises (a) contacting the fusion proteinsof the invention with an inorganic material capable of mineralizing fora sufficient period of time to allow mineralization of the inorganicmaterial.

In one preferred embodiment, the fusion proteins of the invention areformed into a silk film, foam or sponge prior to deposition of inorganicmaterial (mineralization).

In one embodiment, the inorganic material is capable of forminghydroxyapatite or silica.

In one embodiment, the fiber, film, or sponge further comprises anagent.

In one embodiment, the inorganic coating formed on the fibrous proteincomprises an agent.

In one embodiment, the agent is selected from the group consisting of aprotein, peptide, nucleic acid, PNA, aptamer, antibody or a smallmolecule.

The claimed invention also provides for biomaterial products produced bythe methods of the invention.

BRIEF DESCRIPTION OF FIGURES

FIGS. 1A and 1B show diagrammatic illustrations of fibrous proteindomain-mineralization domain fusion proteins. FIG. 1A shows threeclaimed fusion proteins composed of a spider silk fibrous protein domain((CRGD-15mer) (See (SEQ ID NO: 5)) and a dentin matrix protein 1 (DMP 1)mineralization domain. The fusion proteins contain a mineralizationdomain of either 1) the full length amino acid sequence of DMP 1 (See(SEQ ID NO: 7)), 2) the C-terminal end of DMP1, (See (SEQ ID NO: 6)) ora composite sequence based on DMP1, which contains the collagen bindingdomain 1 (CD 1) of DMP1, two acidic clusters in DMP1 that areresponsible for hydroxyapatite nucleation (pA and pB) and the collagenbinding domain 2 (CD2) of DMP1. FIG. 1B shows a diagram of two fusionproteins, 1) fusion of a spider silk fibrous protein domain (CRGD-15mer(See (SEQ ID NO: 5)) and a bone sialoprotein mineralizing domain (BSP)(See (SEQ ID NO: 8)) and 2) fusion of a spider silk fibrous proteindomain 15-mer (without CRGD linker) (See (SEQ ID NO: 4)) and a bonesialoprotein mineralizing domain (BSP) (See SEQ ID NO: 8).

FIG. 2 shows the sequence of the fusion protein CRGD-15mer-CDMP1 (SEQ IDNO: 9). CRGD-15mer-CDMP1 is a fusion protein of a spider silk fibrousprotein domain ((CRGD-15mer) (See, SEQ ID NO: 5), which is indicated bya dark grey highlight, and the C-terminal end of DMP1 mineralizingdomain (See, SEQ ID NO: 6), which is indicated with light greyhighlight.

FIG. 3 shows the sequence of the fusion protein CRGD-15mer-DMP1 (SEQ IDNO: 10). CRGD-15mer-DMP1 is a fusion protein of spider silk fibrousprotein domain ((CRGD-15mer) (See, SEQ ID NO: 5), which is indicated bya dark grey highlight, and the full length sequence of DMP1 mineralizingdomain (See, SEQ ID NO: 7), which is indicated with light greyhighlight.

FIG. 4 shows the sequence of the fusion protein 15mer-BSP (SEQ ID NO:11). 15mer-BSP is a fusion protein of spider silk fibrous protein domain((15mer) (See, SEQ ID NO: 4), which is indicated by light greyhighlight, and a mineralizing domain of bone sialoprotein (BSP) (SEQ IDNO: 8), which is indicated with dark grey highlight.

FIG. 5 shows the sequence of the fusion protein CRGD-15mer-BSP (SEQ IDNO: 12). CRGD-15mer-BSP a fusion protein of spider silk fibrous proteindomain ((CRGD-15mer) (See, SEQ ID NO: 5), which is indicated by lightgrey highlight, and a mineralizing domain of bone sialoprotein (BSP)(SEQ ID NO: 8), which is indicated with dark grey highlight. A linkersequence between the two domains is also present.

FIGS. 6A to 6C show scanning electron microscope (SEM) images ofrecombinant silk film made from CRGD-15mer-CDMP1, See Example 1. FIG.6A, image of silk film morphology prior to mineralization (HFIP+MeOH).FIG. 6B, image of silk film morphology after one round ofmineralization. FIG. 6C, image of silk film morphology after threerounds of mineralization.

FIGS. 7A to 7B show the sequence of the fusion proteins CRGD15mer-R5(SEQ ID NO: 25) and 15mer-R5 (SEQ ID NO: 25) as described in Example II.The underlined sequence represents the monomeric repeat unit selectedand used in the design of the recombinant proteins based on theconsensus sequence of spidroin1 (Maspl) native sequence of Nephilaclavipes (Accession #P19837).

FIG. 8 shows a grid of SEM of untreated and methanol treated silk filmsformed from the four different genetically engineered silk proteinsCRGD15mer, 15mer, CRGD15mer-R5, and 15mer-R5 as described in Example 2.Images of the control films and the films that underwent silicificationreactions are shown.

FIGS. 9A to 9E show SEM images of untreated and methanol treatedelectropun CRGD15mer-R5 silk fibers before, during and aftersilicification reactions, as described in Example 2. FIG. 9A, untreatedelectrospun CRGD15mer-R5 silk fibers. FIG. 9B, electrospun CRGD15mer-R5silk fibers methanol treated before silification. FIGS. 9C, 9D and 9E,electrospun CRGD15mer-R5 silk fibers during silification at 20 um and 2um scale.

FIGS. 10A to 10B show XPS analysis, as described in Example 2, ofCRGD15mer-R5 and silicified CRGD15mer-R5 on A1 foil and on silicon chipat the characteristic binding energies of (FIG. 10A) 153 eV and (FIG.10B) 102 eV for electrons found in the 2s and 2p3 electron shells of thesilicon atom respectively.

FIG. 11 shows Fourier transform infared spectroscopy (FTIR) analysis ofthe structure of CRGD-15mer-CDMP1 before and after Ca ion binding, asdescribed in Example 1.

FIG. 12 shows FTIR analysis of structure of CRGD-15mer-CDMP1 before andafter treatment with methanol, as described in Example 1.

FIGS. 13(A1) to 13(A5) and 13(B1) to 13(B5) show Scanning ElectronMicroscopy (SEM) surface morphologies of recombinant spider silk filmsafter soaking in 1.5×SBF for various periods of time, as described inExample 1. FIG. 13(A1)-FIG. 13(A5), SS15m-CDMP1 soaked in 1.5×SBF for 0,3, 7, 14 and 21 days: FIG. 13(A1)-FIG. 13(A5) respectively. FIG.13(B1)-FIG. 13(B5), SS15m soaked in 1.5×SBF for 0, 3,7,14 and 21 days:FIG. 13(B1)-FIG. 13(B5) respectively. Scale bars are 2 μm.

FIGS. 14A to 14C show Transmission Electron Microscopy (TEM) images ofcrystals grown on CRGD-15mer-CDMP1 after soaking in 1.5×SBF for 21 days,as described in Example 1. (FIG. 14A) image of flake-like apatite; (FIG.14B) electron diffraction pattern; (FIG. 14C) high-resolution image ofapatite crystal.

DESCRIPTION OF THE INVENTION

Fibrous proteins represent an important category of proteins in biology,forming native structural entities both internally in organisms (e.g.,collagens) and externally (such as spun silk fibers for webs andcocoons). As an example, silk proteins from spiders and insects providea number of valuable materials features. When combined with functionaldomains, then the materials properties available from silks becomesignificantly expanded. In accordance with the present invention,various domains from biology known to promote the formation of inorganicstructures, e.g., promote formation of minerals, have been combined witha fibrous protein domain. Thus, these new fusion (composite) materialsoffer the benefits of the fibrous protein material features (e.g, silks)plus the benefit of the inorganic mineralization domains (e.g.,hardness).

In one embodiment, the present invention provides a fusion polypeptidecomprising a fibrous protein domain and a mineralization domain. Thefusion is used to form an organic-inorganic composite. Theseorganic-inorganic composites can be constructed from the nano- to themacro-scale depending on the size of the fibrous protein fusion domainused. In one embodiment, the composites can also be loaded with othercompounds (e.g., dyes, drugs, enzymes) depending on the goal for thematerials, to further enhance function. This can be achieved duringassembly of the material or during the mineralization step in materialsformation.

The fusion polypeptides of the present invention can be used forproduction of silk biomaterials, e.g., fibers, films, foams and mats.See, WO 03/022909. An all-aqueous process may be used. See, WO03/022909.

In another embodiment, the invention provides a method for forming acomposite material. The method comprises contacting a template formedfrom a fusion polypeptide of the invention with a suitable inorganicmaterial for a sufficient period of time to allow mineralization of theinorganic material thus forming an inorganic coating on the template.

In one embodiment the template is in the form of a fiber, film, orsponge.

In an additional embodiment, growth factors, biological components orothers agents, including therapeutic agents, are incorporated into theinorganic coating. The growth factors, biological components or otheragents, can be incorporated during the formation of the template orduring the crystallization process. Such composites can be used todeliver such components to cells and tissues. The ability to incorporategrowth factors, biological regulators, enzymes, therapeutics, or cellsin the construct of the present invention provides for stabilization ofthese components for long term release and stability, as well as bettercontrol of activity and release.

The products produced by these methods offer new options in theformation of scaffolds for biomaterials, tissue engineering applicationsand drug delivery. While the templates are useful in and of themselves,the ability to form inorganic coatings with controlled thickness leadsto control of mechanical properties (e.g., stiffness) and biologicalinteractions, such as for bone formation. Furthermore, the ability tocontrol these processes allows one to match structural and functionalperformance of scaffolds for specific tissue targets and needs.

In another embodiment, the underlying template (e.g., fusion polypeptideformed into a film etc.) can be removed or etched away to generateporous networks, tubes, or lamellar sheets of inorganic material. Thesematerials are useful directly as biomaterial scaffolds, for control ofcell and tissue growth (e.g., as nerve conduits, bone conduits) and fornonbiological applications (e.g., filtration and separation media,catalysis, decontamination (directly or if filled with appropriatechemical or enzymes), radar chaff, coatings in general, and many relatedneeds, for example, inorganic fillers to toughen materials that can alsobe filled with a second component.

The fusion polypeptides may be created, for example, by chemicalsynthesis, or by creating and translation a polynucleotide in which thepeptide regions are encoded in the desired relationship.

It is to be understood that while the invention has been described inconjunction with the preferred specific embodiments thereof that theforegoing description as well as the examples that follow are intendedto illustrate and not limit the scope of the invention. Other aspects,advantages and modifications within the scope of the invention will beapparent to those skilled in the art to which the invention pertains.

Fibrous Protein Domains

Silks are unique in their self-assembly, formation of robust materialsin the form of fibers, films and 3D matrices, and are polymorphic (thestructure can be controlled among many assembly states). For example,silk fibers are the strongest known fibers and rival even highperformance fibers. They are also effective in resisting compression. In3D porous matrices the compressive resistance exceeds other commonlyused organic polymers as biomaterial matrices. Inorganic domains,including silica and hydroxyapatite are prominent in biological systemsas key inorganic components found associated with proteins. Thesemineral phases dominate skeletal components of most systems.

The fibrous protein domain can include short to long versions, with thesize in part influencing the scale of the composite from the nano to themacro level. For example, the size can range from a single sizedhydrophobic block of 50 amino acids, up to multiple blocks as large asdesired including up to proteins of sizes in the 100,000s of Daltons. Inaddition, the fibrous protein fusion domain could include other proteinssuch as collagens, coiled-coiled leucine zipper proteins, elastins,keratins, actins, and tubulins.

The silk protein suitable for use in the present invention is preferablyfibroin or related proteins (i.e., silks from spiders). The silkwormsilk is obtained, for example, from Bombyx mori. Spider silk may beobtained from Nephila clavipes See, for example, WO 97/08315 and U.S.Pat. No. 5,245,012.

Inorganic Domains/Mineralizing Domains

Mineralizing domains include those that can induce the formation ofhydroxyapatite (Example 1), silica (Example 2), cadmium sulfide, andmagnetite. In one embodiment, a mineralizing domain that can inducehydroxyapatite nucleation is obtained from dentin matrix protein 1 orbone sialoprotein (G. He, T. Dahl, A. Veis and A. George. ConnectiveTissue Res. 2003, 44 (Suppl. 1): 240-245; and G. He, T. Dahl, A. Veisand A. George. Nature materials. 2003, 2(8): 552-558; Stubbs et al. JBone Miner Res. 1997 August; 12(8):1210-22). The full molecule or aminimum portion that can induce mineralization can be used, for example,a 37 amino acid segment from DMP (C-terminus) will induce controlledmineralization of hydroxyapatite. In one example, the mineralizationdomains were fused to a sequence derived from spider silk having thefollowing silk fibrous domain repeated 15 times(SGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGT) (SEQ ID NO: 1) in order to generatefunctional recombinant silks that possess both robust mechanicalproperties of the spider silk and the ability to promote mineralization.The recombinant proteins were cloned in pET21a(+) vector and expressedin E. coli. As demonstrated below, the identities of these recombinantsilks were confirmed by amino acid composition analysis and themineralization study was carried out on the surfaces of the castfunctional recombinant silk films using recombinant protein containingonly spider silk sequence as control. It was demonstrated that thefunctional recombinant silks exhibit an ability to promote thenucleation of hydroxyapatite. The functional recombinant silks (silkfusion proteins) of the present invention have potential application inbiomaterials, tissue engineering, advanced material composites andbiosensors.

Preferably, the mineralizing domain is capable of inducing the formationof hydroxyapatite, silica, cadmium sulfide, magnetite and other metalsalts pending choice of peptide domain utilized.

Preferred mineralizing domains include, for example, dentin matrixprotein 1 (DMP1), bone sialoprotein, and fragments of these proteins,and the 19 amino-acid R5 peptide of the Sil1 protein.

Alternative fibrous proteins and mineralizing domains can also beincluded in this system using the template provided. For example,peptides identified from other native proteins or identified bycombinatorial screening methods can be used. Screening methods caninvolve any mineralization assay known in the art, for example thehydroxyapatite and silicification assays described herein.

Preparation of Fusion Proteins; Vectors, Host Cells and Expression

The fusion proteins containing a mineralization domain and a fibrousprotein domain can be made using standard molecular biology methods wellknown to those skilled in the art (See for example, Sambrook et al.,Molecular Biology: A laboratory Approach, Cold Spring Harbor, N.Y. 1989;Ausubel, et al., Current protocols in Molecular Biology, GreenePublishing, Y, 1995).

In some embodiments, the fusion proteins of the invention are made usinglinker sequences. As used herein, the term “linker sequence” refers to ashort (e.g., about 1-20 amino acids) sequence of amino acids that is notpart of the sequence of either of two polypeptides being joined. Forexample, a linker sequence is attached on its amino-terminal end to onepolypeptide or polypeptide domain and on its carboxyl-terminal end toanother polypeptide or polypeptide domain.

The manipulation of nucleic acids that encode the protein domains in thepresent invention is typically carried out in recombinant vectors.Herein, both phagemid and non-phagemid vectors can be used. As usedherein, vector refers to a discrete element that is used to introduceheterologous DNA into cells for the expression and/or replicationthereof. Methods by which to select or construct and, subsequently, usesuch vectors are well known to one of skill in the art. Numerous vectorsare publicly available, including bacterial plasmids, bacteriophage,artificial chromosomes, episomal vectors and gene expression vectors. Avector of use according to the invention may be selected to accommodatea polypeptide coding sequence of a desired size. A suitable host cell istransformed with the vector after in vitro cloning manipulations. Hostcells may be prokaryotic, such as any of a number of bacterial strains,or may be eukaryotic, such as yeast or other fungal cells, insect oramphibian cells, or mammalian cells including, for example, rodent,simian or human cells. Each vector contains various functionalcomponents, which generally include a cloning (or “polylinker”) site, anorigin of replication and at least one selectable marker gene. If givenvector is an expression vector, it additionally possesses one or more ofthe following: enhancer element, promoter, transcription termination andsignal sequences, each positioned in the vicinity of the cloning site,such that they are operatively linked to the gene encoding a polypeptiderepertoire member according to the invention.

Both cloning and expression vectors generally contain nucleic acidsequences that enable the vector to replicate in one or more selectedhost cells. Typically in cloning vectors, this sequence is one thatenables the vector to replicate independently of the host chromosomalDNA and includes origins of replication or autonomously replicatingsequences. Such sequences are well known for a variety of bacteria,yeast and viruses. For example, the origin of replication from theplasmid pBR322 is suitable for most Gram-negative bacteria, the 2 micronplasmid origin is suitable for yeast, and various viral origins (e.g. SV40, adenovirus) are useful for cloning vectors in mammalian cells.Generally, the origin of replication is not needed for mammalianexpression vectors unless these are used in mammalian cells able toreplicate high levels of DNA, such as COS cells.

Advantageously, a cloning or expression vector may contain a selectiongene also referred to as a selectable marker. This gene encodes aprotein necessary for the survival or growth of transformed host cellsgrown in a selective culture medium. Host cells not transformed with thevector containing the selection gene will therefore not survive in theculture medium. Typical selection genes encode proteins that conferresistance to antibiotics and other toxins, e.g. ampicillin, neomycin,methotrexate or tetracycline, complement auxotrophic deficiencies, orsupply critical nutrients not available in the growth media.

Since the replication of vectors according to the present invention ismost conveniently performed in E. coli (e.g., strain TB1 or TG1), an E.coli-selectable marker, for example, the β-lactamase gene that confersresistance to the antibiotic ampicillin, is of use. These can beobtained from E. coli plasmids, such as pBR322 or a pUC plasmid such aspUC18 or pUC19, or pUC119.

Expression vectors usually contain a promoter that is recognized by thehost organism and is operably linked to the coding sequence of interest.Such a promoter may be inducible or constitutive. The term “operablylinked” refers to a juxtaposition wherein the components described arein a relationship permitting them to function in their intended manner.A control sequence “operably linked” to a coding sequence is ligated insuch a way that expression of the coding sequence is achieved underconditions compatible with the control sequences.

Promoters suitable for use with prokaryotic hosts include, for example,the β-lactamase and lactose promoter systems, alkaline phosphatase, thetryptophan (trp) promoter system and hybrid promoters such as the tacpromoter. Promoters for use in bacterial systems will also generallycontain a Shine-Delgarno sequence operably linked to the codingsequence. Preferred promoters for use in the present invention are theisopropylthiogalactoside (IPTG)-regulatable promoters.

In one preferred embodiment of the invention, the fusion protein of theinvention further comprises a tag, e.g. Flag, His, Myc, HA, VSV, or V5,to aid in purification of the protein by standard means. Afterpurification, the fusion protein can be lyophilized or suspended inaqueous solution for preparation of silk materials, such as films, foamsor fibers.

Formation of Silk Fibers, Films, Foams and Gels Using the Silk FusionProtein of the Invention.

The silk fusion proteins of the invention can be processed into films,foams or fibers. As used herein, “silk fibroin” or “silk protein” refersto a silk fusion protein of the invention.

Fibers can be formed by electrospinning Electrospinning can be performedby any means known in the art (see, for example, U.S. Pat. No.6,110,590). Preferably, a steel capillary tube with a 1.0 mm internaldiameter tip is mounted on an adjustable, electrically insulated stand.Preferably, the capillary tube is maintained at a high electricpotential and mounted in the parallel plate geometry. The capillary tubeis preferably connected to a syringe filled with silk/biocompatiblepolymer solution. Preferably, a constant volume flow rate is maintainedusing a syringe pump, set to keep the solution at the tip of the tubewithout dripping. The electric potential, solution flow rate, and thedistance between the capillary tip and the collection screen areadjusted so that a stable jet is obtained. Dry or wet fibers arecollected by varying the distance between the capillary tip and thecollection screen.

A collection screen suitable for collecting silk fibers can be a wiremesh, a polymeric mesh, or a water bath. Alternatively and preferably,the collection screen is an aluminum foil. The aluminum foil can becoated with Teflon fluid to make peeling off the silk fibers easier. Oneskilled in the art will be able to readily select other means ofcollecting the fiber solution as it travels through the electric field.As is described in more detail in the Examples section below, theelectric potential difference between the capillary tip and the aluminumfoil counter electrode is, preferably, gradually increased to about 12kV, however, one skilled in the art should be able to adjust theelectric potential to achieve suitable jet stream.

The process of the present invention may further comprise steps ofimmersing the spun fiber into an alcohol/water solution to inducecrystallization of silk. The composition of alcohol/water solution ispreferably 90/10 (v/v). The alcohol is preferably methanol, ethanol,isopropyl alcohol (2-propanol) or n-butanol. Methanol is most preferred.Additionally, the process may further comprise the step of washing thefibroin fiber in water.

In another embodiment, the biomaterial is a film. The process forforming the film comprises, for example, the steps of (a) preparing anaqueous silk fibroin solution comprising silk protein; (b) adding abiocompatible polymer to the aqueous solution; (c) drying the mixture;and (d) contacting the dried mixture with an alcohol (preferred alcoholsare listed above) and water solution to crystallize a silk blend film.Preferably, the biocompatible polymer is poly(ethylene oxide) (PEO). Theprocess for producing the film may further include step (e) of drawingor mono-axially stretching the resulting silk blend film to alter orenhance its mechanical properties. The stretching of a silk blend filminduces molecular alignment in the fiber structure of the film andthereby improves the mechanical properties of the film.

In a further embodiment, the biomaterial is a foam. Foams may be madefrom methods known in the art, including, for example, freeze-drying andgas foaming in which water is the solvent or nitrogen or other gas isthe blowing agent, respectively.

In one embodiment, the foam is a micropatterned foam. Micropatternedfoams can be prepared using, for example, the method set forth in U.S.Pat. No. 6,423,252, the disclosure of which is incorporated herein byreference.

Formation of Organic-Inorganic Composites

The claimed invention provides a method for forming an organic-inorganiccomposite material. The method comprises contacting a template (such asrecombinant silk films, sponges or fibers) formed from a fusionpolypeptide of the invention with a suitable inorganic material for asufficient period of time to allow mineralization of the inorganicmaterial. Mineralized material is deposited onto the silk forming aninorganic coating on the template.

The procedure for mineralization of the coating is dependent upon themineralization domain that is part of the fusion polypeptide of theinvention. For example, the mineralizing domain of dentin matrix protein1 (DMP1), an acidic phosphoprotein secreted into the extracellularmatrix during the formation and mineralization of bone and dentin, isinvolved in the precipitation of hydroxyapatite, the main inorganiccomponent in calcified hard tissue such as bone and teeth of vertebrates(Koutsopoulos, S. J Biomed Mater Res. 2002, 62: 600-612). Further, themineralizing domain of Silaffin protein is involved in the precipitationof silica.

Those in the art are skilled to select the appropriate buffer forprecipitation of inorganic material. The formation of inorganic coatingsis further described in Examples 1 and 2.

The biomaterial products produced by the processes of the presentinvention may be used in a variety of medical applications such as woundclosure systems, including vascular wound repair devices, hemostaticdressings, patches and glues, sutures, drug delivery and in tissueengineering applications, such as, for example, scaffolding, ligamentprosthetic devices and in products for long-term or bio-degradableimplantation into the human body. A preferred tissue engineered scaffoldis a non-woven network of electrospun fibers.

Additionally, these biomaterials can be used for organ repairreplacement or regeneration strategies that may benefit from theseunique scaffolds, including but are not limited to, spine disc, cranialtissue, dura, nerve tissue, liver, pancreas, kidney, bladder, spleen,cardiac muscle, skeletal muscle, tendons, ligaments and breast tissues.

In another embodiment of the present invention, silk biomaterials cancontain therapeutic agents. To form these materials, the polymer wouldbe mixed with a therapeutic agent prior to forming the material orloaded into the material after it is formed. In addition, the agent canbe incorporated into the inorganic coating formed by the silk fusionprotein mineralization domain by mixing it with the mineralizationsolution. The variety of different therapeutic agents that can be usedin conjunction with the biomaterials of the present invention is vast.In general, therapeutic agents which may be administered via thepharmaceutical compositions of the invention include, withoutlimitation: antiinfectives such as antibiotics and antiviral agents;chemotherapeutic agents (i.e. anticancer agents); anti-rejection agents;analgesics and analgesic combinations; anti-inflammatory agents;hormones such as steroids; growth factors (bone morphogenic proteins(i.e. BMP's 1-7), bone morphogenic-like proteins (i.e. GFD-5, GFD-7 andGFD-8), epidermal growth factor (EGF), fibroblast growth factor (i.e.FGF 1-9), platelet derived growth factor (PDGF), insulin like growthfactor (IGF-I and IGF-II), transforming growth factors (i.e.TGF-.beta.I-III), vascular endothelial growth factor (VEGF)); and othernaturally derived or genetically engineered proteins, polysaccharides,glycoproteins, or lipoproteins. These growth factors are described inThe Cellular and Molecular Basis of Bone Formation and Repair by VickiRosen and R. Scott Thies, published by R. G. Landes Company herebyincorporated herein by reference.

Silk biomaterials containing bioactive materials may be formulated bymixing one or more therapeutic agents with the polymer used to make thematerial. Alternatively, a therapeutic agent could be coated on to thematerial preferably with a pharmaceutically acceptable carrier. Anypharmaceutical carrier can be used that does not dissolve the foam. Thetherapeutic agents, may be present as a liquid, a finely divided solid,or any other appropriate physical form. Typically, but optionally, thematrix will include one or more additives, such as diluents, carriers,excipients, stabilizers or the like.

The amount of therapeutic agent will depend on the particular drug beingemployed and medical condition being treated. Typically, the amount ofdrug represents about 0.001 percent to about 70 percent, more typicallyabout 0.001 percent to about 50 percent, most typically about 0.001percent to about 20 percent by weight of the material. Upon contact withbody fluids the drug will be released.

The biocompatible polymer may be extracted from the biomaterial prior touse. This is particularly desirable for tissue engineering applications.Extraction of the biocompatible polymer may be accomplished, forexample, by soaking the biomaterial in water prior to use.

The tissue engineering scaffolds biomaterials can be further modifiedafter fabrication. For example, the scaffolds can be coated withbioactive substances that function as receptors or chemoattractors for adesired population of cells. The coating can be applied throughabsorption or chemical bonding.

Additives suitable for use with the present invention includebiologically or pharmaceutically active compounds. Examples ofbiologically active compounds include cell attachment mediators, such asthe peptide containing variations of the “RGD” integrin binding sequenceknown to affect cellular attachment, biologically active ligands, andsubstances that enhance or exclude particular varieties of cellular ortissue ingrowth. Such substances include, for example, osteoinductivesubstances, such as bone morphogenic proteins (BMP), epidermal growthfactor (EGF), fibroblast growth factor (FGF), platelet-derived growthfactor (PDGF), vascular endothelial growth factor (VEGF), insulin-likegrowth factor (IGF-I and II), TGF-and the like.

The scaffolds are shaped into articles for tissue engineering and tissueguided regeneration applications, including reconstructive surgery. Thestructure of the scaffold allows generous cellular ingrowth, eliminatingthe need for cellular preseeding. The scaffolds may also be molded toform external scaffolding for the support of in vitro culturing of cellsfor the creation of external support organs.

The scaffold functions to mimic the extracellular matrices (ECM) of thebody. The scaffold serves as both a physical support and an adhesivesubstrate for isolated cells during in vitro culture and subsequentimplantation. As the transplanted cell populations grow and the cellsfunction normally, they begin to secrete their own ECM support.

In the reconstruction of structural tissues like cartilage and bone,tissue shape is integral to function, requiring the molding of thescaffold into articles of varying thickness and shape. Any crevices,apertures or refinements desired in the three-dimensional structure canbe created by removing portions of the matrix with scissors, a scalpel,a laser beam or any other cutting instrument. Scaffold applicationsinclude the regeneration of tissues such as nervous, musculoskeletal,cartilaginous, tendenous, hepatic, pancreatic, ocular, integumenary,arteriovenous, urinary or any other tissue forming solid or holloworgans.

The scaffold may also be used in transplantation as a matrix fordissociated cells, e.g., chondrocytes or hepatocytes, to create athree-dimensional tissue or organ. Any type of cell can be added to thescaffold for culturing and possible implantation, including cells of themuscular and skeletal systems, such as chondrocytes, fibroblasts, musclecells and osteocytes, parenchymal cells such as hepatocytes, pancreaticcells (including Islet cells), cells of intestinal origin, and othercells such as nerve cells, bone marrow cells, skin cells and stem cells,and combination thereof, either as obtained from donors, fromestablished cell culture lines, or even before or after geneticengineering. Pieces of tissue can also be used, which may provide anumber of different cell types in the same structure.

The cells are obtained from a suitable donor, or the patient into whichthey are to be implanted, dissociated using standard techniques andseeded onto and into the scaffold. In vitro culturing optionally may beperformed prior to implantation. Alternatively, the scaffold isimplanted, allowed to vascularize, then cells are injected into thescaffold. Methods and reagents for culturing cells in vitro andimplantation of a tissue scaffold are known to those skilled in the art.

The biomaterials of the claimed invention may be sterilized usingconventional sterilization process such as radiation based sterilization(i.e. gamma-ray), chemical based sterilization (ethylene oxide) or otherappropriate procedures. Preferably the sterilization process will bewith ethylene oxide at a temperature between 52-55° C. for a time of 8hours or less. After sterilization the biomaterials may be packaged inan appropriate sterilize moisture resistant package for shipment and usein hospitals and other health care facilities.

The invention will be further characterized by the following exampleswhich are intended to be exemplary of the invention.

EXAMPLES Example 1 Formation of Hydroxyapatite Silk Fusions

Hydroxyapatite (HAP, Ca₅(PO₄)₃(OH)) is the most stable calcium phosphatesalt at normal temperature and pH between 4 and 12 (Aaron, S. PosnerPhysiological review, 1969, 49(4): 760-792). Hydroxyapatite is the maininorganic component in calcified hard tissue such as bone and teeth ofvertebrates (Koutsopoulos, S. J Biomed Mater Res. 2002, 62: 600-612). Inaddition, hydroxyapatite also has many applications in proteinchromatography, water treatment processes, fertilizer and pharmaceuticalproducts (Bailliez, S.; Nzihou, A.; Beche, E.; Flamant, G. ProcessSafety and Environmental Protection, 2004, 82(B2): 175-180). Forcalcified hard tissue, hydroxyapatite contributes to its stiffness,while organic matrix contributes to its plasticity (Landis, W. J. Bone,16(5): 533-544). The inherent strength and other mechanical propertiesof the skeletal system are thought to depend on an interaction betweenits organic and inorganic matrix strength, here hydroxyapatite. Oneexample is the composite formed between the normal mineral salt,hydroxyapatite, and collagen, the principal organic component of thevertebrate skeleton, able to resist a wide range of compressive ortensile forces whereas either material alone can not (Chen, Q. Z.; Wong,C. T.; Lu, W. W.; Cheung, K. M. C.; Leong, J. C. Y. and Luk, K. D. K.Biomaterials, 2004, 25: 4243-4254). In the present disclosure, wedemonstrate a proof of concept with dental matrix protein (DMP).

Dentin matrix protein 1 (DMP1) is an acidic phosphoprotein secreted intothe extracellular matrix during the formation and mineralization of boneand dentin, therefore, it is believed that DMP 1 plays an important rolein the initiation of mineralization (J. Q. Feng, H. Huang, Y. Lu, L. Ye,Y. Xie, T. w. Tsutsui, T. Kunieda, T. Castranio, G. Scott, L. B.Bonewald, and Y. Mishina. J Dent Res. 2003, 82(10): 776-780; W. T.Butler, H. Ritchie. Int J Dev Biol, 1995, 39: 169-179; George, B.Sabsay, P.A.L. Simonian, and A. Veis. J Biol Chem, 1993,268(17):12624-12630). It has been reported that DMP1 nucleates theformation of hydroxyapatite in vitro (G. He, T. Dahl, A. Veis and A.George. Connective Tissue Res. 2003, 44 (Suppl. 1): 240-245; and G. He,T. Dahl, A. Veis and A. George. Nature materials. 2003, 2(8): 552-558)by binding calcium ions and initiating mineral deposition. It has alsobeen reported that DMP 1 binds to the N-telopeptide region of type Icollagen and the collagen-binding domains involved in this interactionhave been identified (G. He and A. George. J Biol. Chem. 2004, 279(12):11649-11656).

We generated various fibrous protein domain-mineralizing domain fusionproteins. See for example FIGS. 1-5. FIG. 2 shows the sequence ofCRGD-15mer-CDMP1, a fusion protein of a spider silk fibrous proteindomain ((CRGD-15mer) (See, SEQ ID NO: 5), which is indicated by a darkgrey highlight, and the C-terminal end of DMP 1 mineralizing domain(See, SEQ ID NO: 6), which is indicated with light grey highlight.

FIG. 3 shows the sequence of the fusion protein CRGD-15mer-DMP1 (SEQ IDNO: 10). CRGD-15mer-DMP1 is a fusion protein of spider silk fibrousprotein domain ((CRGD-15mer) (See, SEQ ID NO: 5), which is indicated bya dark grey highlight, and the full length sequence of DMP1 mineralizingdomain (See, SEQ ID NO: 7), which is indicated with light greyhighlight.

FIG. 4 shows the sequence of the fusion protein 15mer-BSP (SEQ ID NO:11). 15mer-BSP is a fusion protein of spider silk fibrous protein domain((15mer) (See, SEQ ID NO: 4), which is indicated by light greyhighlight, and a mineralizing domain of bone sialoprotein (BSP) (SEQ IDNO: 8), which is indicated with dark grey highlight.

FIG. 5 shows the sequence of the fusion protein CRGD-15mer-BSP (SEQ IDNO: 12). CRGD-15mer-BSP a fusion protein of spider silk fibrous proteindomain ((CRGD-15mer) (See, SEQ ID NO: 5), which is indicated by lightgrey highlight, and a mineralizing domain of bone sialoprotein (BSP)(SEQ ID NO: 8), which is indicated with dark grey highlight.

Construction of Expression Vector for Recombinant Spider Silks:

The consensus repeat unit of spider silk (-SGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGT-) (SEQ ID NO: 1) was derived from the native sequence of thespidroin 1 of N. clavipes (accession P19837). The construction of theexpression vector pET30a (+) containing 15 repeats of the silk waspreviously described (Bini et al., 2005) and was termed pET30a(+)-15mer. Plasmid containing the rat dentin matrix protein 1 cDNA(pGEX-DMP1) was previously described (Feng et al., J. Dent. Res. 2003,82(10):776-780). Primers with BamHI and XhoI restriction enzyme siteswere designed in order to copy the C-terminal portion of rat DMP 1 frompGEX-DMP 1 by PCR: BamH I site, C-DMP1f:5′-CAGGATCCAGGGGTGACAACCCAGAT-3′ (SEQ ID NO: 26) and Xho I site,C-DMP1r: 5′-GCCTCGAGGTAGCCATCTTGGCAATC-3′ (SEQ ID NO: 27).

PCR products were double digested with BamHI and XhoI before running ona 1% agarose gel. The band of C-terminal DMP1 DNA were cut from gel andpurified using a MinElute gel extraction kit. Expression vector pET21a(+) was also double digested with BamHI and XhoI before running on a0.8% agarose gel. The band of linearized pET21a (+) was purified using aQIAquick gel extraction kit. After determining the DNA concentration ofthe vector pET21a (+) and insert of C-terminal DMP1 based on intensitiesof 1 μl of each in a 1% gel against 1 μl of high mass DNA ladder, theligation reactions were performed using a Quick ligation Kit™ based onan insert to vector molar ratio of 5:1. Fifty μl of one Shot® Mach1™ T1phage-resistant cells were transformed with 1 μl of ligation reaction.The correct clone of vector pET21a (+) containing C-terminal DMP 1,named as pET21a (+)-CDMP 1 was determined by DNA sequencing with forwardand reverse T7 primers. pET21a (+)-CDMP1 was digested with BamHI anddephosphorylated with CIP before running on 0.8% agarose gel. Thelinearized vector was purified using a QIAquick gel extraction kit.pET30a (+)-15mer was digested with BamHI before running on 1% agarosegel and the DNA encoding the repeat unit of spider silk was purifiedusing a MinElute gel extraction kit. The DNA concentrations of thevector pET21a (+)-CDMP1 and the insert: spider silk 15 mer weredetermined using the same method as described above. The spider silk 15mer was then inserted into pET21a (+)-CDMP1 at the BamHI site byligation reaction and the resulting vector was named as pET21a(+)-SS15m-CDMP1. DNA sequencing with forward and reverse T7 primers wasperformed to check the sequences of the vector.

Protein Expression and Purification:

Expression plasmid pET21a (+)-SS15m-CDMP1 was transformed intoEscherichia coli RY-3041 strain, kindly provided by Professor Ry Young(Texas A&M University, College Station, Tex.). High cell densitycultivation using a 1.25 liter jar fermentor (Bioflo 3000; New BrunswickScientific Co., Edison, N.J.) was performed as described previously(Butler et al. Int. J. Dev. Biol. 1995, 39: 169-179). Expression wasinduced by adding isopropyl-β-D-thiogalactoside to a final concentrationof 2 mM at the early log phase when A₆₀₀ was about 30. Cells wereharvested after 3 h of induction by centrifuging the culture at 4° C.,6000×g, for 10 min. The cells were then sonicated on ice using sonicatorequipped with a microtip. Ten second burst at 100 W with a 10 secondcooling period between each burst was applied. The lysate wascentrifuged at 10,000×g for 30 min at 4° C. to pellet the cellulardebris. The recombinant silk protein was purified using Ni-NTA agaroseresin from supernatant at 4° C. The purification fractions that containthe target protein were dialyzed against distilled water for 2 days anddry proteins were prepared by lyophilizing the aqueous solution.Dialyzed purification fractions were also concentrated and desalted byusing Centricon plus 70 (NMW=10 kDa).

Protein Characterization:

SDS-PAGE was performed to analyze purification fractions and Westernblot was performed using His-tag monoclonal antibody to further confirmthe expression of the target proteins. Western blot analysis ofCRGD-15mer-CDMP1 showed expression of the protein (data not shown). Thetarget protein bands on 4-12% Bis-Tris PAGE gel were analyzed at theYale University W. M. Keck Biotechnology Resource Laboratory todetermine amino acid compositions. A Bruker Proflex™ mass spectrometer(Bruker, Billerica, Mass.) was used for molecular weight determination.

Change of Protein Secondary Structure by Calcium Ion Binding:

0.2 mg/mL protein aqueous solution was prepared by the method describedin protein expression and purification. Calcium chloride solution (1M)was added such that a molar ratio of protein to Ca ion was maintained at1:1000. Protein secondary structures before and after adding Ca ion werestudied by Fourier Transform Infrared Spectroscopy (FTIR). FTIR studieswere performed using a Bruker Tensor 27 FTIR spectrometer with a BioATRaccessory.

Film Formation:

Recombinant proteins were dissolved in hexafluoroisopropanol (HFIP) to afinal concentration of 2% w/v. One-hundred μl of silk HFIP solution wasloaded onto a silica wafer and dried in hood. Dried silk film wastreated with 90% methanol to induce the transition of secondarystructure from random coil to beta sheet. Concentrated silk aqueoussolution was then loaded onto silk film and incubated at 37° C.overnight to dry.

In vitro Mineralization:

The silk films formed on silica wafer were then incubated in 1.5×simulated body fluid (SBF). 1.5×SBF was prepared as described previously(3) by dissolving reagent grade CaCl₂, KH₂PO₄, NaCl, KCl, MgCl₂.6H₂O,NaHCO₃, Na₂SO₄ in distilled water and buffering at pH 7.3 withtris-hydroxymethyl aminomethane and hydrochloric acid (HCl). A 1.5×SBFsolution contains 1.5 times higher ion concentration than the SBFsolution with ion concentrations close to human blood plasma. Fresh1.5×SBF was prepared daily to replace the 1.5×SBF. After the silk coatedsilica wafers were soaked in 1.5×SBF for 3, 7, 14 and 21 days, they wereremoved, gently rinsed with distilled water and dried at roomtemperature.

Scanning Electron Microscopy:

Morphological investigation of the dried films before and afterincubating in 1.5×SBF was performed using LEO 982 Field EmissionScanning Electron Microscope (SEM) (LEO Electron Microscopy, Inc.,Thornwood, N.Y.) at 3.0 kV. The sample was sputter coated with goldprior to examination.

Transmission Electron Microscopy:

Samples for transmission electron microscopy (TEM) analysis wereprepared by ultrasonicating of the silk films after incubation in1.5×SBF in 200 μL of absolute ethanol for 10 min and then depositing afew drops of the suspension onto a standard TEM copper grid with a holeycarbon support film. TEM images were obtained with a JOEL 2100 TEMoperating at 200 kV with a LaB₆ filament and recorded with a slow scanCCD camera. The diffraction patterns were obtained at calibrated cameralengths using a NiO_(x) test specimen as a reference.

Results

Protein Expression and Purification:

CRGD-15mer, molecular weight of which is 48.56 kDa, migrated to theright position on SDS-PAGE gel, while the apparent molecular weight ofCRGD-15mer-CDMP1 indicated by SDS-PAGE electrophoresis was higher thanthe calculated molecular weight, which is 58.89 kDa. This is due to highcontent of acidic amino acids such as aspartic acid and glutamic acid inCRGD-15mer-CDMP1. Western blot analysis of CRGD-15mer-CDMP1 andCRGD-15mer-CDMP1 was detected based on the His tag (data not shown).

Amino Acid Composition Analysis:

Amino acid composition analysis confirmed the correct composition forthe purified CRGD-15mer-CDMP1.

Functional Change of Protein Secondary Structure by Calcium Ion Binding:

CRGD-15mer-CDMP1 was unordered random coil in water before adding CaCl₂,indicated by amide I peak at 1649 cm⁻¹ and amide II peak at 1548 cm⁻¹.After Ca ion binding, a helix and β sheet structures showed up,demonstrated by amide I peak at 1659 cm⁻¹ (α helix) and 1632 cm⁻¹ (βsheet) (FIG. 11).

Film Formation:

Silk secondary structure changed from random coil to 13 sheet aftertreated with 90% methanol (FIG. 12) so that the film was more robust insolution.

Functional Assessments of the Fusion Proteins

The apatite nucleation ability of the functional recombinant silks wasstudied using the method of alternate soaking process. Silk films of therecombinant fusion proteins were cast using standard methods.Lyophilized fusion proteins were dissolved in HFIP solvent at aconcentration of 2.5% w/v overnight at 4° C. The silk-HFIP solutionswere then pipetted into 24-well culture plates and left in the hood forabout 3 hours for the silk films to form and dry out. The silk filmswere then treated with 90% v/v methanol to induce β-sheet formation andprevent resolubilization of the films in aqueous solutions.

Scanning Electron Microscopy:

To asses the ability for mineralization, the cast recombinant silk filmwas first soaked in 200 mM aqueous calcium chloride solution bufferedwith tris(hydroxymethyl) aminomethane and HCl (pH 7.4) (Ca solution) for1 hr at 37° C., then Ca solution was aspirated and the film was washedwith abundant distilled water to wash way any unbound Ca²⁺, then 120 mMaqueous disodium hydrogenphosphate (P solution) was added to immerse thesilk film for 1 hr at 37° C. After soaking in P solution for 1 hr, thefilm was washed again by distilled water. This is one round ofmineralization. Three rounds of the soaking were carried out. The filmsurface was then observed by SEM. See FIGS. 6A-6C, which show mineraldeposition on silk film cast using CRGD-15mer-CDMP1. Deposition began tooccur as in round one (FIG. 6B) and continued through round three (FIG.6C). Control films made from CRGD-15mer and 15mer showed no deposition(data not shown). The SEM images indicate that the functional fusionsilk proteins are capable of inducing apatite nucleation and growth,while silk proteins that so not contain mineralizing domains do notpromote apatite nucleation.

In addition, scanning electron microscopy (SEM) surface morphologies ofrecombinant spider silk films after soaking in 1.5×SBF for variousperiods of time were assessed see FIG. 13. FIG. 13(A1)-FIG. 13(A5),SS15m-CDMP1 soaked in 1.5×SBF for 0, 3, 7, 14 and 21 days: FIG.13(A1)-FIG. 13(A5) respectively. FIG. 13(B1)-FIG. 13(B5), SS15m soakedin 1.5×SBF for 0,3,7,14 and 21 days: FIG. 13(B1)-FIG. 13(B5)respectively.

The surfaces of the films made of CRGD-15mer-CDMP1 and CRGD-15mer weresmooth after casting and there was no big difference between two films(FIG. 13A1 and FIG. 13B1). However, after immersing in 1.5×SBF for 3days, the surface morphologies of the films were different (FIG. 13A2and FIG. 13B2).

Transmission Electron Microscopy:

The high-magnification image of the crystals grew on CRGD-15mer-CDMP1(FIG. 14A) showed that the apatite, which appeared flake-like in theSEM, is composed of aggregates of nanocrystals with need shapes 100-200nm in length. The electron diffraction pattern of the nanocrystals (FIG.14B) showed diffraction rings. The spacings of the rings agreed with thecharacteristic x-ray diffraction spacing of hydroxyapatite (JCPDS09-0432). The rings could be assigned to the (002), (211) planes. Inaddition, a high-resolution TEM image (FIG. 14C) showed the latticefringes of apatite nanocrystals. The average distance between fringes is0.82 nm, which is consistent with the value of teh (100) inerplanarspacing in the apatite structure (0.817 nm).

Example 2 Formation of Silica Silk Fusions Introduction

Silica is widespread in biological systems and serves differentfunctions, among which the most essential ones include support andprotection in single-celled organisms, such as diatoms, to higher plantsand animals. Despite its widespread occurrence and importance offunction, little is known about biosilica and the mechanisms used toproduce controlled microscopic and macroscopic silica structures withsuch nanoscale precision and symmetry. The remarkable control in vivo ofthe morphology of these beautiful intricate patterns at small lengthscales are species-specific and has attracted a great deal of interestin recent years as these features exceed the capabilities of present-daysynthetic and technological approaches to materials engineering invitro.

Silaffins^(1;2;3;4;5) form part of three families of proteins identifiedto date in the organic matrix of the cell wall of the diatomCylindrotheca fusiformis. Silaffins are highly post-translationallymodified peptides and have received the most attention because of theirability to induce and regulate silica precipitation at ambienttemperature and pressure. Silaffins are low-molecular weightpolypeptides: natSil-1A (6.5 kDa)⁴, natSil-1B (10 kDa)⁵, and natSil-2(40 kDa)⁵, isolated by treating the diatom cell wall with ammoniumfluoride. A gene sil 1 has been isolated from a C. fusiformis genomiclibrary and it encodes a polypeptide of 265 amino acids. Seven repeatedsequences were identified in this sequence and named R1 to R7¹. R1corresponds to the precursor of Silaffin-1B, while R3 to R7 and R2correspond to the precursors of the Silaffin-1A1 and Silaffin-1A2,respectively.

Controlled formation of biosilica structures by different proteins andpeptides under various physical reaction environments has also beenreported^(6;7;8;9). The 19 amino-acid R5 peptide of the Sil1 protein wasutilized to obtain silica nanostructures with different morphologiesincluding spheres, arch-shaped morphologies and even elongated fibers'.These results suggest that through careful manipulation of theenvironmental conditions and the application of a linear shear force,distinct morphologies can be attained. These opportunities for controlof materials outcomes in biosilica morphology establish important linksto device fabrication from these materials with regularity in processand outcomes to achieve technological relevance in the future, such asfor silica based micro- and nano-devices.

Applicants have genetically cloned the R5 peptide unit of Sil1 proteinto a 15mer spider silk clone of the consensus sequence of the golden orbspider Nephila clavipes, expressed the fusion protein and performed thesame silicification reactions on silk films made from the expressedprotein. The purpose of fusing this silicification-inducing peptide unitto our genetically engineered silk is to combine the properties of oursilk whether in the form of films or other such as spun fibers to thesilica precipitating properties of R5 under ambient conditions toproduce biomaterials with controlled silica morphologies on the surface.These biomaterials are especially useful in such fields as controlleddrug delivery.

Materials and Methods Design and Cloning of Spider Silk Sequences

The repeat unit was selected used in the design of the 15mer andCRGD15mer was based on the consensus sequence of spidroin1 nativesequence of Nephila clavipes (accession P19837)(-SGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGT-) (SEQ ID NO: 1). Multimers encodingthe repeat were cloned through the transfer of cloned inserts betweentwo shuttle vectors based on pUC19 and pCR-Script (Novagen, Wis.), whichwere ampicillin and chloramphenicol resistant respectively^(10;11).

The expression vector pET-30a (Novagen, Madison, Wis.) was modified witha linker carrying the SpeI site flanked by sequences encoding the aminoacids CRGD (SEQ ID NO: 2) to obtain pET30-link. The two complementaryoligonucleotide sequences for the linker were:GGATCCTGTCGCGGTGACACTAGTCGCGGTGACTGTG (SEQ ID NO: 13) andGGATCCACAGTCACCGCGACTAGTGTCACCGCGACAG (SEQ ID NO: 14). The restrictionsites of BamHI and SpeI are underlined. The 15mer sequence obtained bymultimerization was inserted into pET30-link to generatepET30-CRGD15mer. For the production of a spider silk protein withoutCRGD (SEQ ID NO: 2), the construct pET30-15mer was obtained bysubcloning the NcoI-NotI fragment of pCR-15 into pET-30a vector.

To construct the fusion protein CRGD15mer-R5, the polylinker ofpET-21a(+) shuttle vector was redesigned to contain a His-Tag and theCRGD15mer was digested with the restriction enzyme BamHI from pET-30avector and inserted into the pET-21a(+)-link vector. Two complementaryoligonucleotide sequences for the R5 peptide were designed with EcoRIand NotI sites at the 5′ and 3′ ends respectively:

(SEQ ID NO: 15) 5′ aattcagcagcaaaaaaagcggcagctattcgggcagcaaaggcagcaaacgccgcatcctcgc 3′ and (SEQ ID NO: 16) 3′gtcgtcgtttttttcgccgtcgataagcccgtcgtttccgtcgtt tgcggcgtaggagcgccgg 5′

These synthetic oligonucleotides were annealed and ligated into thepET-21a(+)-link vector right next to the CRGD15mer clone to create thefusion protein with the His-Tag at the C-terminus.

To construct the fusion protein 15mer-R5, the synthetic oligonucleotideswere ligated into the NotI and XhoI restriction sites of pET-21a(+)vector right next to the 15mer clone. This fusion protein has a His-Tagat the N-terminus. The amino acid sequence of the R5 peptide of Sil1protein is: SSKKSGSYSGSKGSKRRIL (SEQ ID NO: 17). Use of other Sil1protein sequences are also contemplated, for example, the R2 peptideSSKKSGSYSGYSTKKSGSRRIL (SEQ ID NO: 18), the R3 peptideSSKKSGSYSGYSKGSKRRIL (SEQ ID NO: 19), the R4/R6 peptideSSKKSGSYSGYSKGSKRRNL (SEQ ID NO: 20), the R1 peptide SSKKSGSYYSYGTKK(SEQ ID NO:21).

Protein Expression and Purification

The constructs pET-21(a)+−15mer-R5 and pET-30a-CRGD15mer-R5 were used totransform the E. coli host strains RY-3041, a mutant strain defective inthe expression of SlyD protein, for expression^(12;13). Cells werecultivated in LB broth at 37° C. Protein expression was induced by theaddition of 1 mM IPTG (Fisher Scientific, Hampton, N.H.) when the OD₆₀₀was between 0.6 and 0.8. After approximately 6 hours of proteinexpression, the cells were harvested by centrifugation at 9500 rpm. Forlarge scale expression, E. coli was grown in a fermentor (Bioflo 3000,New Brunswick Scientific Co., Edison, N.J.) in minimal mediumsupplemented with 1% yeast extract. Ammonia was used as the base tomaintain the pH at 6.8. When the pH exceeded 6.88, as a result ofglucose exhaustion in the culture, a feed solution (50% glucose, 10%Yeast Extract, 2% MgSO₄.7H₂O) was added. Pure O₂ was also provided tothe culture to sustain the level of dissolved oxygen above 40%. Allculture media contained kanamycin (50 μg/ml) or ampicillin (100 μg/ml)for selectivity. For the fermentor grown cells, expression was inducedwhen the absorbance was between 25 and 30 at OD₆₀₀.

The cell pellets were resuspended by adding denaturing buffer (100 mMNaH₂PO₄, 10 mM Tris HCl, 8 M urea, pH 8.0) containing 10 mM imidazole.The cells were lysed by stirring for 30 min and were then centrifuged at9500 rpm at 4° C. for 30 min. His-tag purification of the proteins wasperformed by addition of Ni-NTA agarose resin (Qiagen, Valencia, Calif.)to the supernatant (batch purification) under denaturing conditions.After washing the column with denaturing buffer at pH 6.3, the proteinswere eluted with denaturing buffer at pH 4.5 (without imidazole).

SDS-polyacrylamide gel electrophoresis (PAGE) was performed using 4-12%precast NuPage Bis-Tris gels (Invitrogen, Carlsbad, Calif.).Electrophoresis was performed in MOPS buffer for 50 min at 200V.Purified samples were extensively dialyzed against several changes ofH₂O. For dialysis, Snake Skin membranes (Pierce, Rockford, Ill.) withMWCO of 7000 or lower were used. The dialyzed samples were lyophilizedusing a LabConco lyophilizer. For determination of the amino acidcomposition, the samples were submitted to the W. M. Keck FoundationBiotechnology Resource Laboratory (Yale University, New Haven, Conn.).The samples were analyzed from bands of interest cut out from the gel orlyophilized powder after purification and dialysis. Determination ofprotein concentration was performed by BCA assay (Pierce, Rockford,Ill.) or the molar absorptivity at 280 nm. FIGS. 7A and 7B show thesequence of CRGD15mer-R5 (FIG. 7A) and 15mer-R5 (FIG. 7B)

Silicification Reactions

The lyophilized fusion proteins were then dissolved in HFIP solvent at aconcentration of 2.5% w/v overnight at 4° C. The silk-HFIP solutionswere then pipetted into 24-well culture plates and left in the hood forabout 3 hours for the silk films to form and dry out. The silk filmswere then either left as is or treated with 90% v/v methanol to induceβ-sheet formation and prevent resolubilization of the films in aqueoussolutions. 100 mM phosphate buffer at pH 5.5 was added to the silk filmsand leave to sit for about 30 minutes before 1M tetramethoxysilane(TMOS) dissolved in 1 mM hydrochloric acid was added to the mixture forabout 10 minutes. The films were then washed with MilliQ water threetimes and left to dry overnight in the fume hood. These films were thenanalyzed using a LEO 982 Scanning Electron Microscope at the CIMSfacility at Harvard University.

SEM Results

In order to exploit the self-assembling properties of silk in developingsilk-silica nanocomposites, experiments were performed using TMOS aloneas the precursor. Four genetically engineered variants of the spidersilk protein (two controls, one with and one without RGD but bothwithout R5, two chimeric versions of the controls but with R5) were castinto films that were either left untreated or were treated with methanolto induce a structural transition to β-sheet and thus decrease filmsolubility in aqueous buffer. Silicification reactions were performed onthe films yielding spherical silica structures with diameters rangingfrom ˜0.5 to 2.0 um only when the silica precipitating domain, R5peptide, was fused to the C-terminus of the silk proteins (FIG. 8). Thesilk proteins that did not contain R5, CRGD15mer and 15mer, did notyield significant changes in surface morphology of the films uponexposure to the silicification reactions.

Fusion proteins were assembled into fibers by electrospinning. SEMimages of the electropun fibers formed from the chimeric proteins (FIGS.9A-9D), and the morphological characteristics observed when the fiberswere treated with methanol, were similar to those we observed previouslyfor electrospun silk fibroin with polyethylene oxide¹⁵. Uponsilicification on electrospun mats formed from the chimeric proteinCRGD15mer+R5 without methanol treatment, similar spherical silicastructures were observed as in the reactions on the cast films. However,the dimensions of the silica spheres were slightly smaller, ranging from200 to 400 nm (FIG. 9). When the electrospun fibers consisting of thechimera CRGD15mer+R5 were not treated with methanol, the fibers fusedtogether on the surface. Without the β-sheet inducing methanoltreatment, the fibers are prone to partially solubilize on the surfaceyielding a thin film upon which the mineralization reaction takes place.However, upon treatment of the chimera CRGD15mer+R5 electrospun matswith methanol before silicification, a thin film formed from thesolubilized and then fused fibers at the surface and silica nanospheresdid not form as in the sample above. Instead, the fibers fused with eachother as expected¹⁵ and although mineralization occurred as confirmed byXPS, silica deposited around the fibers providing a non-uniform coatinginstead of the spheres (FIG. 9). When the chimera CRGD15mer+R5 waselectrospun during the silica polymerization process (concurrentprocessing), silica deposition was induced in and on the fibers andelliptically shaped silica particles fused to the fibers were observed(FIG. 9). XPS analysis of the resulting fibers confirmed the presence ofelemental silicon (FIG. 10). Thus, the concurrent processing approach,fiber spinning and silicification reactions, resulted in a differentmorphology of the silica in terms of location within the fibers andshape, compared to the silicification reactions conducted postelectrospinning.

SEQUENCES

Fibrous protein domain sequence derived from Spidroin1 (the nativesequence of the goldon orb spider Nephilia clavipes)<

(SEQ ID NO: 1) SGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGT

A linker sequence CRGD (SEQ ID NO: 2)

The consensus fibrous protein domain sequence derived from Spidroin1(the native sequence of the goldon orb spider Nephilia clavipes) withCRGD linker

(SEQ ID NO: 3) CRGDTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGT

15 mer: An amino acid sequence that contains a repeat of a fibrousprotein domain sequence derived from Spidroin1 (the native sequence ofthe goldon orb spider Nephilia clavipes) The fibrous protein domainsequence is repeated 15 times.

(SEQ ID NO: 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 

CRGD-15mer: an amino acid sequence that contains a repeat of a fibrousprotein domain sequence derived from Spidroin1 (the native sequence ofthe goldon orb spider Nephilia clavipes) The fibrous protein domainsequence is repeated 15 times with a CRGD linking sequence

(SEQ ID NO: 5) MASMTGGQQMGRGSCRGDTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSRGDCGSHHHHHH 

CDMP1, the C-Terminal Sequence of DMP1, a Mineralizing domain

(SEQ ID NO: 6) RGDNPDNTSQTGDQRDSESSEEDRLNTFSSSESQSTEEQGDSESNESLSLSEESQESAQDEDSSSQEGLQSQSASRESRSQESQSEEDSRSEENRDSDSQDSSRSKEESNSTGSTSSSEEDNHPKNIEADNRKLIVDAYHNKPIGDQ DDNDCQDGY

DMP1, the full length sequence of DMP1, a Mineralizing domain

(SEQ ID NO: 7) LPVARYQNTESESSEERTGNLAQSPPPPMANSDHTDSSESGEELGSDRSQYRPAGGLSKSAGMDADKEEDEDDSGDDTFGDEDNGPGPEERQWGGPSRLDSDEDSADTTQSSEDSTSQENSAQDTPSDSKDHHSDEADSRPEAGDSTQDSESEEYRVGGGSEGESSHGDGSEFDDEGMQSDDPGSTRSDRGHTRMSSADISSEESKGDHEPTSTQDSDDSQDVEFSSRKSFRRSRVSEEDDRGELADSNSRETQSVSTEDFRSKEESRSETQEDTAETQSQEDSPEGQDPSSESSEEAGEPSQESSSESQEGVASESRGDNPDNTSQTGDQRDSESSEEDRLNTFSSSESQSTEEQGDSESNESLSLSEESQESAQDEDSSSQEGLQSQSASRESRSQESQSEEDSRSEENRDSDSQDSSRSKEESNSTGSTSSSEEDNHPKNIEADNRKLIVDAYHNKPIGDQDDNDCQDGY 

BSP: Bone sialoprotein, a Mineralizing domain

(SEQ ID NO: 8) SEFPVQSSSDSSEENGNGDSSEEEEEEEENSNEEENNEENED SDGNED

CRGD-15mer-CDMP1: (SEQ ID NO: 9): a fusion protein of (SEQ ID NO: 5) and(SEQ ID NO: 6)<

(SEQ ID NO: 9) MASMTGGQQMGRCRGDTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSRGDCGSRGDNPDNTSQTGDQRDSESSEEDRLNTFSSSESQSTEEQGDSESNESLSLSEESQESAQDEDSSSQEGLQSQSASRESRSQESQSEEDSRSEENRDSDSQDSSRSKEESNSTGSTSSSEEDNHPKNIEADNRKLIVDAYHNKPIGDQDDNDCQDGY 

CRGD-15mer-DMP1 (SEQ ID NO: 10): a fusion protein of (SEQ ID NO: 5) andSEQ ID NO: 7)<

(SEQ ID NO: 10) MASMTGGQQMGRCRGDTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSRGDCGSLPVARYQNTESESSEERTGNLAQSPPPPMANSDHTDSSESGEELGSDRSQYRPAGGLSKSAGMDADKEEDEDDSGDDTFGDEDNGPGPEERQWGGPSRLDSDEDSADTTQSSEDSTSQENSAQDTPSDSKDHHSDEADSRPEAGDSTQDSESEEYRVGGGSEGESSHGDGSEFDDEGMQSDDPGSTRSDRGHTRMSSADISSEESKGDHEPTSTQDSDDSQDVEFSSRKSFRRSRVSEEDDRGELADSNSRETQSVSTEDFRSKEESRSETQEDTAETQSQEDSPEGQDPSSESSEEAGEPSQESSSESQEGVASESRGDNPDNTSQTGDQRDSESSEEDRLNTFSSSESQSTEEQGDSESNESLSLSEESQESAQDEDSSSQEGLQSQSASRESRSQESQSEEDSRSEENRDSDSQDSSRSKEESNSTGSTSSSEEDNHPKNIEADNRKLIVDAYHNKPIGDQDDNDCQDGY

15mer-BSP (SEQ ID NO: 11): a fusion protein of (SEQ ID NO: 4) and (SEQID NO: 8)

(SEQ ID NO: 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 

CRGD-15mer-BSP (SEQ ID NO: 12): a fusion protein of (SEQ ID NO: 5) and(SEQ ID NO: 8) with linker

(SEQ ID NO: 12) MASMTGGQQMGRGSCRGDTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGOGAGAAAAAGGAGOGGYGGTGSOGTSGRGGTGGOGAGAAAAAGGAGOGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGTSRGDCGSESEFPVQSSSDSSEENGNGDSSEEEEEEEENSNEEENNEENEDSDGNEDKLHHHHHH 

One of two complementary oligonucleotide sequences for the linkercarrying the SpeI site flanked by sequences encoding the amino acidsCRGD to obtain pET30-link

(SEQ ID NO: 13) GGATCCTGTCGCGGTGACACTAGTCGCGGTGACTGTG 

One of two complementary oligonucleotide sequences for the linkercarrying the SpeI site flanked by sequences encoding the amino acidsCRGD to obtain pET30-link

(SEQ ID NO: 14) GGATCCACAGTCACCGCGACTAGTGTCACCGCGACAG 

One of two complementary oligonucleotide sequences for the R5 peptide(contains the 19 amino-acid R5 peptide of the Sil1 protein) which weredesigned with EcoRI and NotI sites at the 5′ and 3′ ends respectively

(SEQ ID NO: 15) 5′ aattcagcagcaaaaaaagcggcagctattcgggcagcaaaggcagcaaacgccgcatcctcgc 3′

One of two complementary oligonucleotide sequences for the R5 peptide(contains the 19 amino-acid R5 peptide of the Sil1 protein) which weredesigned with EcoRI and NotI sites at the 5′ and 3′ ends respectively.

(SEQ ID NO: 16) 3′ gtcgtcgtttttttcgccgtcgataagcccgtcgtttccgtcgtttgcggcgtaggagcgccgg 5′

The amino acid sequence of the R5 peptide of Sil1 protein from C.fusiformis is: SSKKSGSYSGSKGSKRRIL (SEQ ID NO: 17)

The amino acid sequence of the R2 peptide of Sil1 protein from C.fusiformis is: SSKKSGSYSGYSTKKSGSRRIL (SEQ ID NO: 18),

The amino acid sequence of the R3 peptide of Sil1 protein from C.fusiformis is: SSKKSGSYSGYSKGSKRRIL (SEQ ID NO: 19),

The amino acid sequence of the R4/R6 peptide of Sil1 protein from C.fusiformis is: SSKKSGSYSGYSKGSKRRNL (SEQ ID NO: 20).

The amino acid sequence of the R1 peptide of Sil1 protein from C.fusiformis is: SSKKSGSYYSYGTKK (SEQ ID NO:21).

Bone Sialoprotein Precursor [Homo sapiens] Accession AAC95490 is:

(SEQ ID NO: 22) MKTALILLSI LGMACAFSMK NLHRRVKIED SEENGVFKYR PRYYLYKHAY FYPHLKRFPV QGSSDSSEEN GDDSSEEEEE EEETSNEGEN NEESNEDEDS EAENTTLSAT TLGYGEDATPGTGYTGLAAI QLPKKAGDIT NKATKEKESD EEEEEEEEGNENEESEAEVD ENEQGINGTS TNSTEAENGN GSSGGDNGEEGEEESVTGAN AEGTTETGGQ GKGTSKTTTS PNGGFEPTTP PQVYRTTSPP FGKTTTVEYE GEYEYTGVNE YDNGYEIYES ENGEPRGDNY RAYEDEYSYF KGQGYDGYDG QNYYHHQ 

Silaffin 1 precursor (natSil-1) [Contains: Silaffin-1B; Silaffin-1A2;Silaffin-1A1] Accession Q9SE35: MKLTAIFPLL FTAVGYCAAQ SIADLAAANLSTEDSKSAQL ISADSSDDAS DSSVESVDAA SSDVSGSSVE SVDVSGSSLE SVDVSGSSLESVDDSSEDSE EEELRILSSK KSGSYYSYGT KKSGSYSGYS TKKSASRRIL SSKKSGSYSGYSTKKSGSRR ILSSKKSGSY SGSKGSKRR1 LSSKKSGSYS GSKGSKRRNL SSKKSGSYSGSKGSKRRILS SKKSGSYSGS KGSKRRNLSS KKSGSYSGSK GSKRRILSGG LRGSM (SEQ ID NO:23)

CRGD15mer-R5: Sequence of fusion protein; fusion of CRGD15mer to theamino acid sequence of the R5 peptide of Sil1 protein from C.fusiformis.

(SEQ ID NO: 24) MASMTGGQQM GRGSCRGDTS GRGGLGGQGA GAAAAAGGAGQGGYGGLGSQ GTSGRGGLGG QGAGAAAAAG GAGQGGYGGLGSQGTSGRGG LGGQGAGAAA AAGGAGQGGY GGLGSQGTSGRGGLGGQGAG AAAAAGGAGQ GGYGGLGSQG TSGRGGLGGQGAGAAAAAGG AGQGGYGGLG SQGTSGRGGL GGQGAGAAAAAGGAGQGGYG GLGSQGTSGR GGLGGQGAGA AAAAGGAGQGGYGGLGSQGT SGRGGLGGQG AGAAAAAGGA GQGGYGGLGS QGTSGRGGLG GQGAGAAAAA GGAGQGGYGG LGSQGTSGRG GLGGQGAGAA AAAGGAGQGG YGGLGSQGTS GRGGLGGQGA GAAAAAGGAG QGGYGGLGSQ GTSGRGGLGG QGAGAAAAAG GAGQGGYGGL GSQGTSGRGG LGGQGAGAAA AAGGAGQGGYGGLGSQGTSG RGGLGGQGAG AAAAAGGAGQ GGYGGLGSQGTSGRGGLGGQ GAGAAAAAGG AGQGGYGGLG SQGTSRGDCGSEFSSKKSGS YSGSKGSKRR ILCGRHHHHH H

15mer-R5: Sequence of fusion protein; fusion of 15mer to the amino acidsequence of the R5 peptide of Sil1 protein from C. fusiformis.MHHHHHHSSG LVPRGSGMKE TAAAKFERQH MDSPDLGTDD DDKAMASGRG GLGGQGAGAAAAAGGAGQGG YGGLGSQGTS GRGGLGGQGA GAAAAAGGAG QGGYGGLGSQ GTSGRGGLGGQGAGAAAAAG GAGQGGYGGL GSQGTSGRGG LGGQGAGAAA AAGGAGQGGY GGLGSQGTSGRGGLGGQGAG AAAAAGGAGQ GGYGGLGSQG TSGRGGLGGQ GAGAAAAAGG AGQGGYGGLGSQGTSGRGGL GGQGAGAAAA AGGAGQGGYG GLGSQGTSGR GGLGGQGAGA AAAAGGAGQGGYGGLGSQGT SGRGGLGGQG AGAAAAAGGA GQGGYGGLGS QGTSGRGGLG GQGAGAAAAAGGAGQGGYGG LGSQGTSGRG GLGGQGAGAA AAAGGAGQGG YGGLGSQGTS GRGGLGGQGAGAAAAAGGAG QGGYGGLGSQ GTSGRGGLGG QGAGAAAAAG GAGQGGYGGL GSQGTSGRGGLGGQGAGAAA AAGGAGQGGY GGLGSQGTSG RGGLGGQGAG AAAAAGGAGQ GGYGGLGSQGTSSSKKSGSY SGSKGSKRRI L (SEQ ID NO: 25)

PCR PRIMER BamH I site, C-DMPlf: 5′-CAGGATCCAGGGGTGACAACCCAGAT-3′ (SEQID NO: 26)

PCR PRIMER Xho I site, C-DMP1r: 5′ GCCTCGAGGTAGCCATCTTGGCAATC-3′ (SEQ IDNO: 27).

The references cited herein and throughout the application areincorporated by reference.

REFERENCES

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What is claimed is:
 1. An organic-inorganic composite materialcomprising: (i) a fusion polypeptide comprising a fibrous protein domainand a mineralizing domain, wherein the mineralizing domain is capable ofinducing mineralization; and (ii) an inorganic material.
 2. Theorganic-inorganic composite material of claim 1, wherein the fibrousprotein domain is obtained from silk, collagens, coiled-coiled leucinezipper proteins, elastins, keratins, actins, or tubulins.
 3. Theorganic-inorganic composite material of claim 1, wherein the fibrousprotein domain comprises an amino acid sequence from the silk proteinSpidroin
 1. 4. The organic-inorganic composite material of claim 1,wherein the fibrous protein domain comprises an amino acid sequence asset forth in SEQ ID NO: 1 or SEQ ID NO:
 3. 5. The organic-inorganiccomposite material of claim 4, wherein the fibrous protein domaincomprises an amino acid sequence as set forth in SEQ ID NO: 4 or SEQ IDNO:
 5. 6. The organic-inorganic composite material of claim 1, whereinthe fusion polypeptide is crystallized.
 7. The organic-inorganiccomposite material of claim 1, wherein the mineralizing domain inducesthe formation of hydroxyapatite, silica, cadmium sulfide or magnetite.8. The organic-inorganic composite material of claim 7, wherein themineralization domain is derived from dentin matrix protein 1 (DMP1),bone sialoprotein (BSP), silaffin-1 (Sil1) protein, or functionalfragments thereof.
 9. The organic-inorganic composite material of claim8, wherein the mineralization domain comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 6, 9, and 17-21. 10.The organic-inorganic composite material of claim 9, wherein themineralization domain comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 7, 22, and
 23. 11. The organic-inorganiccomposite material of claim 1, wherein the fusion polypeptide comprisesan amino acid sequence selected from the group consisting of SEQ ID NOs:9-12, 24, and
 25. 12. The organic-inorganic composite material of claim1, wherein the inorganic material is hydroxyapatite or silica.
 13. Theorganic-inorganic composite material of claim 1, wherein theorganic-inorganic composite material further comprises an active agent.14. The organic-inorganic composite material of claim 13, wherein theagent is selected from the group consisting of naturally derived orgenetically engineered proteins, polysaccharides, glycoproteins,lipoproteins, peptides, nucleic acids, PNAs, aptamers, antibodies, smallmolecules, and cells.
 15. The organic-inorganic composite material ofclaim 13, wherein the agent is a therapeutic agent, a cell attachmentmediator, a hormone, a chemoattractant, or a growth factor.
 16. Theorganic-inorganic composite material of claim 15, wherein the cellattachment mediator comprises an integrin binding amino acid sequence.17. The organic-inorganic composite material of claim 15, wherein thetherapeutic agent is selected from the group consisting ofanti-infectives; chemotherapeutic agents; anti-rejection agents;analgesics and analgesic combinations; and anti-inflammatory agents. 18.The organic-inorganic composite material of claim 15, wherein the growthfactor is selected from the group consisting of bone morphogenicproteins, bone morphogenaic-like proteins, epidermal growth factors,fibroblast growth factors, platelet derived growth factor (PDGF),insulin like growth factors, transforming growth factors, and vascularendothelial growth factor (VEGF).
 19. The organic-inorganic compositematerial of claim 1, wherein the organic-inorganic composite material isin a form selected from a fiber, a film, a sponge, a gel, a foam, and amicropatterned foam.
 20. A tissue engineered scaffold comprising anorganic-inorganic composite material of claim 1.